This research program is multiphasic with the major emphasis on the regulation of the biotin operon. The isolation of a purified repressor protein will permit us to carry out a series of physical-chemical studies to determine how the repressor protein functions in the control of the biotin operon. The binding of biotin and biotin analogs and competition studies can best be carried out with the ammonium sulfate precipitation technique or by equilibrium dialysis. The interaction of the biotin-repressor complex wit the operator site/s can be visualized by electron microscopy. The chemical nature of the operator site/s will be determined by base sequence analysis of the DNA fragment convered by the biotin-repressor complex. The stability of the biotin-repressor-operator complex will be determined from kinetic studies on the association and dissociation of the complex using the filter-binding technique. The kinetics of transcription and repression of the biotin operon with purified polymerase and repressor proteins will be studied by the hybridization technique. In addition, we plan to explore a proposed pathway for pimeloyl CoA synthesis with an isotopic tracer technique. For the isolation of the biotin permease protein, we will prepare a lambda lysogen carrying the bioP gene to enhance permease synthesis. The isolation of pleiotropic mutants which are unable to convert dethiobiotin to biotin, may provide evidence for a multi-step reaction.